ES2605705T3 - Tiethylperazine for the treatment of a beta-amyloidopathy or alpha-synucleopathy - Google Patents

Abstract

2- (R2-thio) -10- [3- (4-R1-piperazin-1-yl) propyl] -10H-phenothiazine according to the general formula I ** Formula ** in which the remainder R1 is a methyl group, the R2 moiety is an ethyl group and the R3 and R7 moieties are hydrogen, for the treatment of a β-amyloidopathy or an α-synucleinopathy, which are accompanied by a deposit of brain proteins and a reduced activity of the cerebral ABCC1 transporter .

Description

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DESCRIPTION

Tiethylperazine for the treatment of a beta-amyloidopathy or alpha-synucleopathy

The accumulation of proteins or fragments of proteins (peptides) in the brain is a significant feature of age-related neurodegenerative diseases. In the case of Alzheimer's dementia (Alzheimer's disease, AD) and cerebral p-amyloid angiopathy (CAA), the aggregation of p-amyloid peptides (Ap) is triggering the disease, the underlying mechanism being unknown. Ap-proteostasia, that is the balance of production and degradation / transport by means of receptors or proteases, is altered in the case of AD and CAA. However, the separation of Ap peptides by cellular transporters (ABC transporters) has so far been given little attention. In the case of Parkinson's disease (Parkinson's disease) the a-synuclema protein accumulates, which among other things regulates the distribution of dopamine in the black substance. In the case of Parkinson's disease a-synucleinopathy it is known that ABC transporters play a decisive role for transport (Kortekaas et al., Ann Neurol 2005, 57, 176179). According to this, there are several AG subfamilies, which can transport different substrates in a bidirectional way (metabolites, drugs, peptides, proteins, ions) and can even be changed redprocally in the transport function (for example ABCB1 and ABCC1, Tao et al. Cancer Chemotherapy and Pharmacology, 64, 5, 961-969).

WO2008 / 092898A1 describes a combination of two active ingredients for the treatment of diseases that occur through the addition of proteins. In the case of one of the active substances, it is a derivative of phenothiazine, not including thiethylperazine. WO96 / 04915A1 describes the treatment of Alzheimer's disease by means of derivatives of phenothiazine or thioxanthenes, not including thiethylperazine. WO2008 / 133884A2 describes the treatment of a plurality of neurodegenerative diseases with a plurality of active ingredients. In the case of one of the active substances it is a question of thiethylperazine. However, all experimental data refer to Huntington's disease.

By means of different mouse models modified by genetic engineering it could be shown that the ABC conveyor (the joint structural element of the ABC conveyors is an ATP binding cassette and a transport pore), ABCC1, is an important protectant / peptide transporter , particularly Ap transporter, which has extraordinary functional effects on the accumulation of brain proteins. ABCC1 is also an important a-synuclema transporter.

The following studies describe the activity of transporters by way of example in the transport of Ap.

In order to determine the activity of ABCC1 in vivo, the genetically engineered in GM mice expressing APP in each case was separated by the ABCB1 transporter, the ABCG2 transporter or the ABCC1 transporter (knockout mice).

In this regard it was found that:

i) the amount of Ap in mice lacking the ABCC1 transporter had increased 12 times,

ii) loss of ABCB1 transporter leads only to a 3-fold increase and

iii) the loss of ABCG2 has no effect of accumulation of Ap.

Therefore, it was an objective of the present invention to provide substances that adequately influence the ABCC1 transporter so as to be able to treat neurodegenerative diseases, in particular p-amyloidopaths or a-synucleinopaths. This objective was solved with 2- (R2-thio) -10- [3- (4-R1-piperazin-1-yl) propyl] -10H-phenothiazine according to claim 1. Other preferred embodiments result from the dependent claims.

In other words, the objective was solved by 2- (R2-thio) -10- [3- (4- R1-piperazin-1-yl) propyl] -10H-phenothiazine according to general formula I

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image 1

wherein the R moiety is a methyl group, the R2 moiety is an ethyl group and the R3 and R7 moieties are hydrogen, for the treatment of a p-amyloidopathy or an-synucleinopathy, which are accompanied by a deposit of brain proteins and a reduced activity of the cerebral ABCCl transporter.

There is also a need for possibilities to recognize or diagnose or predict these diseases in the case of a-synucleinopathies and in the case of p-amyloidopathies.

The facilitation of a procedure was also objective of the descriptive memory, with which a-synucleinopathies as well as p-amyloidopathies could be diagnosed or prediagnosed. This objective is solved by means of a procedure for the diagnosis or prediagnostic of a p-amyloidopathy or a-synucleinopathy or for the determination of the risk of an experimental person suffering from such a disease, in which the experimental person already takes substances which are transported through the cerebral ABCC1 transporter, which is constituted by the following stages:

a) determination of the amount of the substance ingested in samples of body fluids of the person from

experimentation at a specific time;

b) repetition of the determination of stage a) at least another subsequent time;

c) comparison of the quantities determined in step a) and b) with quantities that had been defined in

equal moments as characteristics for people of experimentation, which at the time of extraction

of the samples showed no clinical indication of a p-amyloidopathy or a-synucleinopathy.

That the experimental person already takes at least one substance that is transported through the cerebral ABCC1 transporter means that this substance should not be administered in the first place. Rather, this already exists in the body of the experimental person, for example due to a pharmacological treatment of another disease. The samples of body fluids of the experimental person, which are subject to study, are preferably samples of blood plasma, blood serum and / or cerebrospinal fluid.

P-amyloidopathy is preferably an Alzheimer's dementia, a-synucleinopathy is preferably Parkinson's disease. Eventually it can be a-synucleinopathy also a dementia by Lewy bodies (DLB). Substances that are transported through the cerebral ABCC1 transporter are preferably selected from antibiotics (for example difloxacin, grepafloxacin), virostatic / antiviral drugs (for example saquinavir, ritonavir), anti-allergy / antihistammics (for example cimetidine), cardiovascular drugs (for example verapamil), antidepressants (for example citalopram), antihyperuricemic agents (for example probenecid), cytostatics (for example methotrexate, etoposit, edatrexate, ZD1694), vitamins / analogs of vitamins (for example methotrexate, folic acid, L-leucovorin), antiflogistic for example indomethacin), antiepileptics (for example valproic acid), hormones / hormone derivatives (for example 17p-estradiol), leukotrienes (for example LTC4), fluorescent samples (for example calcema, Fluo-3, BCECF, SNARF), metabolites of natural substances (produced endogenously) coupled with GSH, sulfate or glucuronide, toxins or drugs (for example 2,4-dinitrof enil-SG, bimane-SG, N-ethylmaleimide-SG, doxorubicin-SG, thiotepa-SG, cyclophosphamide-SG, melfalan-SG, chlorambucil-SG, ethacrynic acid-SG, metolachlor-SG, atrazine-SG, sulforafan-SG , aflatoxin B1-epoxide-SG, 4- nitroquinoline 1-oxido-SG, As (SG) 3, ethoposid-Gluc, 4- (methylnitrosamino) -1- (3-pyridyl) -1-butanol (NNAL) -3p- O-Gluc, SN-38-gluc, 4-methylumbelliferyl-pd-gluc, 6-hydroxy-5,7-dimethyl-2-methylamino-4- (3-pyridylmethyl) -benzothiazole (E3040S) -Gluc, leukotriene C4, prostaglandin A2-SG, 15-deoxy-A12,14 prostaglandin J2-SG, hydroxynonenal-SG, 17p-estradiol-17-pd-gluc, glucuronosylbilirubin, bis-lucuronosylbilirubin, hydrodeoxycholate-6-a-Gluc, estrone-3 - sulfate, dehydroepiandrosterone sulfate, sulfatolitocolato) (see also Deeley RG et al .: Substrate recognition and transport by multi drug resistance protein 1 (ABCC1), FEBS letters 2006, 580 (4), p. 1103-1111.)

This indirect analysis of the transporter activity of the ABCC1 transporter can be used for the diagnosis / prediagnostic of a corresponding disease. In experimental people who already take substances that can be transported by ABCC1 in other ways, the profile of the concentration of active substance in body fluids, preferably blood plasma, blood serum and / or serum may be studied.

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Cerebrospinal fluid. A temporarily dependent measurement shows for people of experimentation, in which there is a reduced ABCC1 transporter activity compared to healthy experimentation people, a delayed or displaced substance concentration curve (concentration c plotted with respect to time t), that is the maximum of the curve appears temporarily modified.

When a displaced curve results in comparison with the healthy case, this is an indication of a modified activity of the ABCC1 transporter. This means that then also substances such as Ap or a-synuclema are transported worse and is therefore an indication of a corresponding disease.

Both the mouse models and also the pharmacological influence of ABCC1 show that this is an important cellular transmembrane transporter for the Ap protein and implies that the blood-brain barrier and the choroid plexus occupy a key position for the excretion of Ap out of the brain. It could be shown that selective pharmacological activation of the ABCC1 transporter significantly reduces brain load with Ap and can thus be used therapeutically for the treatment of diseases with altered cerebral proteostasis. In addition, the analysis of the transporter activity of the ABCC1 transporter, as described above, can be used for the indirect or direct diagnosis / prediagnostic of a corresponding disease. Direct analysis will be possible through the administration of substances that are transported through the ABCC1 conveyor, and its determination. The indirect analysis was explained earlier.

Modifications of export mechanisms, which are related to ABC transporters, can substantially influence the temporal aggregation profile of Ap and other brain proteins. As a result, an influence of the function of the ABCC1 transporter has a positive effect on the risk of suffering from neurodegenerative diseases, particularly Alzheimer's. The "treatment of neurodegenerative diseases" includes in this sense prophylaxis as well as the treatment of existing diseases.

The role of ABC transporters in the excretion of Ap was first studied so that it was determined that ABCC1 can transport Ap. For this purpose, in vitro Transwell assays with endothelial cells (endothelialcelltranswellassay, ECTA) of primary capillary endothelial cells were used. , grown from mouse brains (cell culture mixture):

Primary cultures of endothelial cells of cerebral capillaries of ABCB1 deficient mice, ABCC1 deficient (knockout) and control mice (C57BI / 6, FVB / N) were used to study the specific transport activity of Ap. Transport of Ap from the abluminal compartment (brain) to the luminal compartment (blood) is altered in endothelium deficient in ABCB1 and deficient in ABCC1. The average speed of transport of Ap during the first six hours after administration of Ap peptides (Ap42) was 2.2 pg / min for control cells. In contrast, ABCC1-deficient cells reached only half of the transport capacity (1.0 pg / min). In ABCB1-deficient cells, the transport of Ap (0.3 pg / min) was barely present. Other studies of capillary endotheliums and choroid plexus cells resulted in the ABCB1 transporter being highly expressed in cerebral capillary endothelies, while the expression of endothelial ABCC1 is lower in cerebral capillaries.

By means of Alzheimer's mouse models deficient in newly generated ABC transporters, the relative significance of members of the ABC transporter family in vivo was then studied. Mice modified by genetic engineering have in each case a deficiency (knock out) of specific ABC transporters ABCG2, ABCB1 or ABCC1.

Ap immunohistochemistry of brain cuts showed:

i) significant increases in cortical number and size of Ap-positive plaques in ABCC1 deficient mice compared to control mice (see Figures 1 and 2a-c).

ii) Mice deficient in ABCB1 showed a lower increase in the number and size of Ap plaques than mice deficient in ABCC1.

iii) No significant difference could be detected between control mice and ABCG2-deficient mice (Figure 2a-c).

For the determination of the amount of soluble Ap in buffer (mostly monomers and smaller oligomers) and of soluble Guanidine Ap (mostly fibrillar or aggregate material) enzyme coupled immunoadsorption assays (Enzyme-linkedimmunoabsorbentassays) were used , ELISAs) for Ap.

According to the morphological results of immunohistochemistry, ABCC1-deficient mice showed a significant increase in case of aggregate Ap compared to control mice at all times of measurement. The brain load with Ap was the most intense at an age of 25 weeks. At this time, Ap (Ap42) values were 12 times higher than in control mice. Ap soluble in buffer also increased

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with age, however after 25 weeks, at the time of the highest plate loading, the values of soluble Ap in the ABCC1-deficient group greatly decreased.

Other studies were conducted that provided other evidence of the relationship between eventually deficient transport through ABCC1 and the aggregation of Ap.

The transport kinetics of ABC transporters depend, among other things, on specific protein / peptide characteristics, such as specific cargo. The Dutch type variant of the amyloid precursor protein (Dutch mutant, APPdt), which introduces an additional negative charge near the cut-off site of APP a-secretase and thus leads to severe cerebral amyloid angiopathy (CAA), influences the elimination of APdt through the blood-brain barrier. Western blot analysis of cerebral capillaries and choroid plexus (CP) of control mice showed a strong expression of ABCB1 in cerebral capillary endotheliums (BC) and ABCC1 in CP (Figure 3d). Since ABC transporters play an important role in the elimination of Ap, it was assumed that mice deficient in ABC, APPdt-transgenic (in the blood-brain barrier and in the blood-brain-choroid plexus barrier) have a reinforced accumulation of APdt in menmotic vessels. The degree of CAA in APPdt mice deficient in ABC was quantified at the age of 24 months. According to the assumption, at least 51% of the vessels (> 75% of the vascular wall loaded with Ap) in ABCC1-deficient animals compared with 23% in the controls were highly altered (Figure 3c).

Based on these results, the extent to which the soluble Ap content in the brain could be reduced / influenced was mediated through activation mediated by active principles of ABC transporters. Mice were treated with amyloid deposits for 30 days with anti-emetic thiethylperazine (Torecan®, 2- (ethylthio) -10- [3- (4- methylpiperazin-1-yl) propyl] -10H-phenothiazine). Twice a day, 3 mg / kg body weight was administered intramuscularly. Prophylactic treatment began before the mice formed senile plaques. ELISA measurements of treated animals showed a reduction in the amount of Ap by at least 31% in mice treated compared to animals treated with vehicle (vehicle = water) (Figure 3e). The results are graphically reproduced in Figure 3.

The elimination capacity of Ap was a key factor in the regulation of the intracerebral accumulation of Ap.

As an especially effective activator of the ABCC1 transporter, it was thiethylperazine (Torecan®). Other derivatives based on the same base structure also showed good results in activating the ABCC1 transporter. The corresponding derivatives are represented in general formula I

image2

in which the remains

R1 and R2 are the same or different and are in each case independently of each other C1-C6 alkyl groups, which independently present with each other another substituent selected from the group alkyl, aryl, acyl (preferably acetyl), amino, nitro, sulfonyl, hydroxyl, alkoxy, aryloxy, arylthio, alkylthio and halogen atom, the respective alkyl groups having at least one other halogen atom, and the R3 moiety is in one of positions 6-9 of the phenothiazine ring system, preferably in position 6 , 7 or 8, and a hydrogen atom or is an alkyl, aryl, acryl (preferably acetyl), amino, nitro, sulfonyl, hydroxyl, alkoxy, aryloxy, arylthio or alkylthio or halogen atom group, having the respective alkyl groups possibly at least one other halogen atom, or is an NR4R5 or OR6 group, R4, R5 and R6 being the same or different and in each case independently selected from each other hydrogen and alkyl group C1- C3 and the rest

R7 is in one of positions 1, 2 or 4 of the phenothiazine ring system, preferably in position 2 or 4, and a hydrogen atom or is an alkyl, aryl, acryl (preferably acetyl), amino, nitro group , sulfonyl, hydroxyl, alkoxy, aryloxy, arylthio or alkylthio or a halogen atom, the respective alkyl groups having at least one other halogen atom, or is an NR8R9 or OR10 group, R8, R9 and

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R10 the same or different and in each case independently selected from s ^ of hydrogen and C1-C3 alkyl group.

These derivatives are correspondingly very suitable for the treatment of neurodegenerative diseases, in particular of p-amyloidopaths or a-synucleinopaths, the treatment as previously mentioned both prophylaxis and the treatment of existing diseases. The halogen atom / halogen atoms are preferably selected from fluorine and chlorine. The acyl groups (- (C = O) -R) of the R 1, 2, 3 7 moieties are preferably acetyl groups (-C (= O) CH 3). Preferably, the R1 and R2 moieties are the same or different and in each case independently from each other a C1-C6 alkyl group or a C1-C6 alkyl group (preferably C1 alkyl) substituted with an acetyl group, as well as the R3 and R7 moieties are hydrogen or an acetyl group. Preferably, the radicals R1 and R2 are the same or different and in each case independently from each other a C1-C3 alkyl group. It is also preferred that the R3 and R7 moieties be hydrogen. It is especially preferred when the R1 moiety is a methyl group, the R2 moiety is an ethyl group and the R3 and R7 moieties are hydrogen (tiethylperazine, Torecan®). In the use for the treatment of neurodegenerative diseases it has been advantageous to add to the 2- (R2-thio) -10- [3- (4-R1-piperazin-1-yl) propyl] -10H-phenothiazine other active ingredients, preferably one-

benzohydrylpiperazines, most preferably 1-benzohydril-4-cinnamyl-piperazine (cinnarizine).

Various neurodegenerative diseases can be treated with the 2- (R2-thio) -10- [3- (4-R1-piperazin-1- yl) propyl] -10H-phenothiazine derivative according to the invention or can be diagnosed by the indirect analyzes described above. In a particularly preferred embodiment, neurodegenerative disease is a p-amyloidopathy, in particular Alzheimer's dementia (AD). Another embodiment refers to the case where neurodegenerative disease is an a-synucleinopathy, in particular Parkinson's disease (PD). Both diseases, that is p-amyloidopathy and a-synucleinopaths, are characterized by deposits of brain proteins that can be treated by means of an activation of the ABCC1 transporter or can be diagnosed through their activity.

Other diseases that can also be treated through the activation of the ABCC1 transporter or can be diagnosed through the activity of the ABCC1 transporter, are mentioned below. Thus is another disease that can be treated dementia by Lewy bodies (LBD). This is also characterized by aggregation of brain proteins, that is, an a-synucleinopathy such as Parkinson's.

Another embodiment not according to the invention refers to the case that the neurodegenerative disease is Huntington's disease (HD). Another embodiment not in accordance with the invention relates to the case that neurodegenerative disease is a disease caused by prions, in particular Creutzfeld-Jacob disease (CJD) or fatal familial insomnia (FFI). Another embodiment not in accordance with the invention refers to the case where the neurodegenerative disease is a tauopathy, in particular cortico-basal degeneration (CBD), Steel-Richardson-Olszewski (PSP, progressive supranuclear palsy-supranuclear paralysis) syndrome or Pick's disease (PiD). Another embodiment not in accordance with the invention refers to the case that the neurodegenerative disease is a frontotemporal degeneration (FTLD), in particular positive degeneration for ubiquitin, positive degeneration for TDP43 or negative degenerations for ubiquitin and TDP43. Another embodiment not according to the invention refers to the case where the neurodegenerative disease is amyotrophic lateral sclerosis (ALS). Another embodiment not according to the invention refers to the case that the neurodegenerative disease is a spinocerebellar ataxia (ACS) or spastic paraparesis (SPG). Another embodiment not according to the invention refers to the case where the neurodegenerative / neuroimmune disease is multiple sclerosis (MS) or an MS-related syndrome, in particular ADEM or Devic's syndrome.

Description of the figures Show

Figure 1a that the cortical density of neuter palcas in ABCC1-deficient mice (ABCC1ko) has been raised by ~ 75%;

Figure 1b, c that the average plate size has risen (+34%) due to the greater number of plates (+63%) with a size of more than 700 | im2 and a lower frequency of smaller plates (- 24%) Error bars, standard error (n> 3);

1d shows that the IHC staining in ABCG2-deficient mice (ABCG2ko), ABCB1-deficient (ABCB1ko), ABCC1-deficient (ABCC1ko) and in control mice shows a higher surface density of Ap in ABCC1-deficient animals. Typical plates of the same size are represented in cut, the scale bars represent 500 | im (top view) and 50 | im (cut) (* p <0.05);

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figure 2a

figure 2b figure 2c

Figure 3 Figure 3a Figure 3b

figure 3c

figure 3d figure 3e

Examples

Animals

that the density of plaques in cortex (cover) and size in mice deficient in specific ABC transporters is high. In particular, ABCC1-deficient mice (ABCC1ko) show a high amyloid load Ap (light gray bars, in each case to the right outside in the individual clusters), w = week on the abscissa axis;

that the total plate size in mice deficient in ABCC1 (ABCCIko) and deficient in ABCB1 (ABCBIko) at the age of 25 weeks is high, w = week on the abscissa axis;

that the total increase in plate size is associated with the appearance of fewer small plates and more larger plates (> 700 | im2), while the number of medium-sized plates remains at the same value, error bars, standard error (n> 5), * p <0.05;

that ABCC1 deficiency promotes the accumulation of Ap and APdt and that the activation of ABCC1 (through Torecan administration) reduces Ap values; in which

shows that at an age of 25 weeks the deficiency in ABCC1 leads to a marked increase (~ 12 times) in insoluble Ap; Y

shows that the amount of soluble Ap42 in buffer at an age of 25 weeks is markedly reduced compared to 22 weeks (-56%). This is probably based on deposition in insoluble deposits. At the same age, the area occupied by depositions of Ap, which is measured in immunohistochemistry, has risen by 83% (error bars, standard error n> 5, p <0.05);

shows that 53% of blood vessels are greatly altered by CAA (> 75% of vascular walls show Ap). This refers to mice deficient in ABCC1 (ABCCIko) compared to 23% in controls (n = 3);

shows that the expression of ABCC1 can be observed predominantly in the choroid plexus (CP), while ABCB1 is expressed mainly in the capillaries of the brain (BP);

shows that the activation of ABCC1 by thiethylperazine (Torecan) reduces Ap values in mice (-28%), error bars, standard error (n = 4, * p <0.05).

APP-transgenic mice (APP, APPdt) were purchased from The Jackson Laboratory (Bar Harbor, USA) and from the University of Tubingen (Tubingen, Germany). NEP-deficient mice were purchased from Riken Brain Research Institute (Saitama, Japan). Mice deficient in ABCG2, ABCB1 and ABCC1 were purchased from Taconic-Farms (Denmark). All transgenic and deficient mouse lines were crossed for at least 9 generations in the FVB gene pool. The mice were kept in a light / dark cycle from 12h / 12h at 23 ° C with free access to food and water.

Procedures

Tissue preparation

For tissue preparation, mice were sacrificed by cervical dislocation and perfused transcardially with PBS (physiological sodium chloride solution, phosphate buffered). The brain was separated and a hemisphere was placed in 4% paraformaldetute buffered for paraffin embedding and immunohistochemistry. The other hemisphere was rapidly frozen in liquid nitrogen and stored at -80 ° C for biochemical analysis.

ELISA

ELISA kits (TH40HS, TK42HS) from The Genetics Company (TGC, Schlieren, Switzerland) were used for the quantification of Ap. The cerebral hemispheres were homogenized using PreCellys24 (12 s, 6,500 rpm). After adding carbonate buffer (pH 8.0), the homogenates were mixed using PreCellys (5 s, 5,000 rpm) and centrifuged for 90 min at 4 ° C and 24,000 g, to separate insoluble and soluble Ap species. The remaining supernatant (buffer soluble fraction) was mixed with 8M guanidine hydrochloride in a ratio of 1: 1.6. For the extraction of the added Ap species, the sediment was dissolved in 8 volumes of 5 M guanidine hydrochloride, stirred at room temperature for 3 h and centrifuged at 24,000 g for 20 min at 4 ° C. The remaining supernatant represented the guanidine soluble fraction (GuaHCl). Protein content was measured three times

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of all samples, using a Nanodrop1000 spectrophotometer (ThermoFisher Scientific, Wilmington, USA). ELISAs were performed according to the manufacturer's instructions using appropriate dilutions.

Western blots

Tissue homogenates were prepared for Western blots. Total protein concentrations of the extracts were determined using a BCA assay (Pierce, Teil von Thermo Fisher Scientific, Rockford, USA). After electrophoresis of 10 µg of total protein per lane, the proteins in PVDF membranes were immunoblotted. After blocking in 5% of milk powder in TBST buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.1% Tween20) for 1 h at room temperature, the immunoblotting was studied or to determine ABCB1 (1: 500, D-11, Santa Cruz), ABCC1 (1: 200, Alexis Bio) or p-actin (1: 20,000, Sigma) overnight at 4 ° C. As detection antibodies were used HRP anti-mouse, HRP anti-rat or HRP anti-hare. For the visualization, an Amersham ECL Plus detection kit and a Roper-CoolSnap HQ2 camera were used.

Immunohistochemistry (IHC)

The formalin-fixed brains were embedded in paraffin and 4 | im thick sections were cut. After the separation of the paraffin, the sections were subsequently treated with a BondMax ™ autotender (Menarini / Leica, Germany). Immunostaining was initiated after blocking endogenous peroxidase (5 min) and reunion of epftopos (epitoperetrieval) for 5 min with 95% formic acid (for 6F3D antibodies, Dako, Germany) and 70% formic acid (for 4G8 antibodies, Millipore, Germany). Primary antibodies were routinely incubated at room temperature for 30 min with the following dilutions: 6F3D (1: 100), 4G8 (1: 500). Primary antibodies were detected with the BondMax ™ Bond Polymer Refine detection kit and according to the standard DAB R30 protocol. The cuts were completely digitized with a resolution of 230 nm using a MiraxDesk / MiraxMidi scanner and then analyzed automatically using the AxioVision software package (Zeiss, Germany).

CAA severity assessment

Brain sections of APPdt mice were stained with 4G8 antibodies. At least two non-consecutive sections were studied to determine CAA of the menmotic vessels blindly. All menmgeous vessels were counted manually and the severity of the CAA was classified as follows:

Category I: not altered

Category II: <25% of the periphery had positively Category III: <50% of the periphery had positively Category IV: <75% of the periphery had positively Category V: <100% of the periphery held positive way

The average number of vessels for any category was calculated with respect to the total number of vessels detected.

Transwell endothelial cell assay (ECTA)

Endothelial cells of mouse brain capillaries were prepared as described in Coisne et al. (Coisne, C. et al. Mouse syngenic in vitro blood-brain barrier model: a new tool to examine inflammatory events in zerebral endothelium. Laboratory Investigation; 85, 734-746 (2005)). At least ten mice 3-4 weeks old were decapitated and the brains were separated. After dissecting the brainstem, the white matter and the meninge, the tissue was homogenized in two volumes of wash buffer B (WBB) (Hanks bufferedsaltsolution (HBBS), 10 mM HEPES, 0.1% BSA) using a Dounce homogenizer 15 ml glass (Wheaton Industries, Millville, NJ; USA). A volume of 30% dextran solution was added to the homogenate. It was centrifuged twice at 3,000 g and 4 ° C. The sediment containing the vessels was resuspended in WBB and the large vessels were manually split by strong pipetting of the solution. Vacuum filtration by 60 | im membranes (SEFAR, Switzerland) was used to separate large vessels from the capillaries. After combined treatment with collagenase / dispase (HBSS, 10 mM HEPES, 0.15 | ig / ml TCLK, 10 | ig / ml DNAsa-I, collagenase / disparate 1 mg / ml (Roche)) a suspension of individual cells was achieved by additional strong pipetting of the solution. Endothelial cells were introduced into Transwell inserts coated with Matrigel (pores of 0.4 | im, Greiner Bio-One, Germany) with a density of 120,000 cells per insert and allowed to grow in a glial support culture.

Sulfur yellow was used for the determination of paracellular flow during the test. The culture medium of the abluminal compartment was replaced with a solution containing 10 ng of Ap42 (final concentration 1.6 nM). Next, samples were taken from the luminal compartment after 2 h, 6 h or 24 h and the Ap content was determined with ELISA (TK42-highsense, TGC, Switzerland). The transport speed was determined as described in Coisne et al. (Coisne, C. et al. Mouse syngenic in vitro blood-brain barrier model: a new tool to examine inflammatory events in cerebral endothelium. Laboratory Investigation; 85, 734-746 (2005)).

ELISA statistics

The Lilliefors goodness of fit test (alpha = 0.05) was applied to ELISA data and ELISA data transformed into log, to differentiate between the assumption of normally distributed sample data and the assumption of data. of samples normally distributed in log. Despite the small sample size, the zero hypothesis (Ho) for 5 of 44 samples was rejected for the two data sets. In accordance with the observation that positive biases (skew) and strictly positive sample data predominate, the assumption of normally distributed data was ruled out. The mean values and confidence intervals were calculated assuming a normal distribution in the underlying log. The Wilcoxon ranks sum test was applied to compare the ELISA data of the different mouse strains for each moment.

Claims (6)

5 10 fifteen twenty 25 1.2- (R2-thio) -10- [3- (4-R1-piperazin-1-yl) propyl] -10H-phenothiazine according to general formula I image 1 in which the radical R1 is a methyl group, the radical R2 is an ethyl group and the radicals R3 and R6 7 are hydrogen, for the treatment of a p-amyloidopathy or an-synucleinopathy, which are accompanied by a deposit of brain proteins and reduced activity of the ABCC1 brain transporter. 2. 2- (ethyl-thio) -10- [3- (4-methyl-piperazin-1-yl) propyl] -10H-phenothiazine for use according to claim 1, characterized in that other active ingredients are added . 3. 2- (ethyl-thio) -10- [3- (4-methyl-piperazin-1-yl) propyl] -10H-phenothiazine for use according to claim 2, characterized in that as other active ingredients add 1-benzohydrilpiperazines. 4. 2- (ethyl-thio) -10- [3- (4-methyl-piperazin-1-yl) propyl] -10H-phenothiazine for use according to claim 2 or 3, characterized in that as 1- Benzohydrilpiperazine is added 1-benzohydril-4-cinnamyl-piperazine. 5. 2- (ethyl-thio) -10- [3- (4-methyl-piperazin-1-yl) propyl] -10H-phenothiazine for use according to one of the claims 1 to 4, characterized in that p-amyloidopathy is Alzheimer's dementia. 6. 2- (ethyl-thio) -10- [3- (4-methyl-piperazin-1-yl) propyl] -10H-phenothiazine for use according to one of the claims 1 to 4, characterized in that a-synucleinopathy is Parkinson's disease or dementia by Lewy bodies.